Oleandomycin, its salts and production



July 31, 1956 Original Filed June 1. 195 3 IfzgJ INFRARED ABSORPTION SPECTRUM OF AMIMYCIN BASE \uvE NUMBERS m cn" 1500 I400 I300 I200 B. A. SOBIN ETAL 2,757,123

OLEANDOMYCIN IT'S SALTS AND PRODUCTION 2 Sheets-Sheet 1 c o c o 2 co 3 N o g INVENTORS BONVHIHSNVUI M30831! BE A 503/ JOHN 8. NUT/Ell BY THOMAS H. LEES TH ll? ATM/"If July 31, 1956 A. SOBIN ETAL 2,757,123

OLEANDOMYCIN ITS SALTS AND PRODUCTION Original Filed June 1. 1953 2 Sheets-Sheet 2 I500 I400 I500 l2(X) INFRARED ABSORPTION SPECTRUM OF AMIMYCIN HYDROCHLORIDE 5 g INVENTORS ZIONVllIHSNVHl 1N30H3d BE A 503/ JOHll I]. BOUT/Ell THOMAS H. LEES B *WM THE ATTWMEY United States Patent OLEANDOMYCIN, ITS SALTS AND PRODUCTION 2,757,123 Patented July 31, 1956 Some of the significantdifierences between the new strain ATCC 11891 and the description of S. antibioticus in Bergeys Manual are described in Table II.

TABLE II Ben A. Sobin, Flushing, N. Y., John Routien, Tenafly, 5

E gii gf g g g g; h i 2 33 3 2 35 53 .55? Medium Strain ATCC 11891 Bergeys Description ration of Delaware Gelatin Gray, waxy myceli- Dark brown growth on sur- Continuation of application Serial No. 358,804, Iune 1, 11111; no sp s g g rggfiw of y 1953- This application June 1955 Sena! N0 10 Litmus milk (In skimmedmilk, Thick, bro wnish ring on sur- 518,722 no litmus.) face of milk. Mouse gray Creamy white to aerialmycelium with green- 9 Claimstrainees un asiness hydrolysis. milk; no clearing. This invention relates to a new and useful antibiotic g ggzgggg g gj 'sqlggl gg gf gg Solublepigmemdeepbmwn called oleandomycin and more particularly, to its producstances. darkbrown. tion by fermentation, to methods for its recovery and concentration from crude solutions, such as fermentation The strain of S. antibioticus described in Bergeys Manbroths, to processes for its purification, and to methods for l Produces h ibi ti ti i hi h f o th the preparation Of its salts. The invention includes Within data given h ereinafter is clearly established to be difierits scope the antibiotic in dilute forms, as crude conm f om oleandomycin, centrates and in pure crystalline forms. These novel I i to be u d d thapfor h production f 1 Products are especially useful in Combaflrng Pathogenic andomycin the present invention'is not limited to this microorganisms, Particularly Gram-Positive microorgaparticular organism or to organisms fully answering the nisms. The present application is a continuation of our 5 above description, which is given only for illustrative Coperldirlg application Serial 358,804 filed June purposes. In fact, it is especially desired and intended to 1953 and now abandoned. include the use of mutants produced from the described The new antibiotic is formed during the cultivation unrganism by ariou means, u h a X-radiation, ultrader controlled conditions, of a new strain of a species of vi l t diatio nitrogen mustards, and the like. microorganism known as Streptomyces-antibioticus, which Oleandomycin shows high activity against a wide variewas identified by planting and testing a culture thereof ty of microorganisms. As previously mentioned, howon media normally used for the identification of such ever, it is particularly noteworthy in its action on Gram- Inicroorganisms. A culture of the microorganism has positive organisms. While it demonstrates some activity been deposited in the American Type Culture c ll cagainst Gram-negative organisms, this activity is generally tion, Washington, D. C., and added to its collection of a somewhat lower level. The following table illusof microorganisms as ATCC 11891. The identification of trates the antibiotic spectrum of oleandomycin against a this new strain, designated Isolate No. 15784-1, in the variety of Gram-negative and Gram-positive microculture collection of Chas. Pfizer & Co., Inc., of Brooklyn, organisms. v These tests were run by seeding nutrient N. Y. was made with the aid of Bergeys Manual of broth containing various concentrations of the pure Determinative Bacteriology, sixth edition (1948). antibiotic with the particular organism specified. The The cultural characteristics of the new strain of S. minimum inhibitory concentration indicated in Table antibioticus are set forth in the following table. Ex- III is the minimum concentration of the antibiotic (in cept where otherwise noted, the results are based upon micrograms/milliliter) at which growth of the microsix replicates incubated for two weeks. The colors where organism failed to occur. Since the highest concentra- R is written are those of Ridgway, Color Standards and tion employed in this test was mcg./ml., the mini- Nomenclature. mum inhibitory concentration is not precisely stated TABLE I Streptomyces antibioticus ATCC No. 11891 Color Medium Amount of Growth Remarks Aerial Mycelium and Sporulation Soluble Pigment Glucose-Asparagine Agar Moderate White aerial mycelium; pooosporw- Medium brown to dark Reverse brown; conidiophores lation. brown. gnficlusltgrs; no spirals; spores 5X n. Gelatin .do Gay, waxy mycelium; no sporula- Dark brown Good liquefaction.

1011. skimmed milk (28 C.) do Creamy white ring No coagulation; hydrolysis. N o

a change in pH. Glucose agar .do Light brown, waxy, transparent Light brown Reverse light brown.

growth; no sporulation. Calcium malate Poor to moderate... White aerial mycelium; sporulation None Reverse creamy white, calcium poor, grayish white. malate digested. Synthetic agar Poor Mouse-gray (R) sporulatlon -do Reverse white to light gray. Nutrient agar do Light tlflrown, waxy, transparent Medium brown Reverse light brown.

grow Emersons agar Poor to moderate". Light gray to yellow, waxy wrinkled Dark brown Do.

mycelium; no sporulation. Cellulose Poor White, aerial mycellum Dextrose nitrate broth. do Nitrates not reduced. Potato discs Moderate Near Dawn Gray (R) sporulation... Starch plates oor Yellow to brown, waxy growth; Reverse dark yellow to gray; no

sporulation near Hathi Gray (R). hydrolysis.

3 where such concentration apparently exceeded 100 mcg./ml. The test was conducted under standardized conditions. i

TABLE III Spectrum of oleandomycin Minimum Inhibitory Organism Concentration Oleanclomycin, mcg./ ml.

Gram-Negative Organisms:

Brucella bronchiseptiea 25 A. aerogenes 2 100 E. coli 21 100 Proteus Sp. 1 100 Ps. aeraginosa 173 100 Salmonella typhasa 344 100 K. pneumoniae 132 100 S. paratyphra A134 100 S. paratyphia B139 100 Listeria monocytogenes... 3.12 Nez'sseria gonorrheae..- I 6. 25 Niesseria catarrhalis- 3. 12 Neissen'a meningitidis- 6. 25 Gram-Positive Organisms:

Strep. faecalis 121 1. 56 Micrococcas pyogenes var. aareus 0.19 Micrococcas pyogenes var. aareaa 209 0. 19 M. pyogeaes var. albas 3-- 0. 78 B sabtilis 2 0.39 6. 25 0.19 3.12 3.12

0.3 0.7 0. 7 CH) 0.5 Cortmebacteriam zeroes. 6. 25 Clastridiam perjringens. 1. 5 Clostrrdtam sporogeaes- 1. 5 Clostridia'm tetani. 1. 5 Bacillus anthracis 7 Fungi:

Candida albicans 8 100 Table IV shows the results of similar tests conducted in another laboratory on different organisms or strains 4 TABLE IV Oleandomycin spectrum, additional microorganisms Minimum Inhibitory Organism Concentration Oleandemycin megJ ml.

Gram-Negative Organisms:

E. coli 225 E. colt 450 E. typhosa 225 Salmonella sp... 450 Aerobacter aerogene 225 S. paratyphosa 900 Klebsiella pneumonia 225 Brucella bronchiscptica 7 900 112 112 225 225 150 225 225 Proteus vulgar 000 Gram-Positive Organi Strep. hemo i 1. 7 D. pneamom'ae 1. 7 Staph. n- 1. 7 B. sabtilis 1. 7 Emer0c0ccus 1. 7 OZ. welchii 7 Staph. aareas 235 1. 7 Fungi:

Mom'lia albicans 000 TABLE V Antibiotic: Minimum Inhibitory Concentration in mcgJnil. M. puoaenes var. aareus Strain Oleando- Oxytetra- Aureo- Chlor- Baoi- Neo- Pem- Poly- I mycin cycline 1 mycin amiphlentracin mycin cillin myxin 0.78 78 0.39 v 3. 12 PI 28 6. 0 3. 75 250 0. 78 100 100 6. 25 14 78 60 250 0. 78 100 50 6. 25 28 3. 12 0. 47 250 PI indicates partial inhibition. I

Terramycm."

I 1 Sold under the trademark the spectrum of oleandomycin- In these tests, themaximum concentration of the antibiotic was 900 mcg./ml.

eiiectiveness of the antibiotic these tests also indicate oxytetracycline,

In addition to showing the against these resistant strains, that the oleandomycin difiers from aureomycin, chloramphenicol, bacitracin, neornycin, penicillin and polymyxin.

Oleandomycin has also been shown to be effective against a variety of mycobacteria, as indicated in Table VI which gives the results of tests performed on such mycobacteria. In this case, tubes of Dubos liquid medium were used as the test medium. The antibiotic was added to the medium at the various levels indicated in the first column of the table. The tubes of medium containing such levels or concentrations of the antibiotic were then seeded with cultures of the various mycobacteria specified, the identity of the species being indicated at the head of each of the columns in the table. The test was then conducted by incubating the tubes under sterile conditions for 36 hours and thereafter observing them to determine the presence or lack of growth of the mycobacteria. Lack of growth in the tubes at the end of the test period is indicated by a sign, and growth is indicated by TABLE VI Activity of Oleandomycin against mycobaczeria Meg/ml. nmae phlez' smegmatis 607 Oleandomycin has been found to possess a relatively low level of toxicity when used in test animals. For example, the LDo value, when the antibiotic is administered intraveneously to mice as a solution in water, is approximately 15 mg./20-gram mouse. Toxicity to other species and by other routes of administration is comparable.

Oleandomycin has also been found to possess a high order of activity in vivo against various pathogenic organisms. Mice of substantially uniform weight were intraperitoneally infected with certain strains of Strep. hemolyticus, D. pneumoniae and M. pyogenes var. aureus and treated with the antibiotic by subcutaneous injection in two mg. doses daily for two and one-half days. The effectiveness of oleandomycin against these organisms in the mice thus treated is readily apparent from Table VII which compares the percent survival of treated and untreated mice.

TABLE VII Activity of Oleandomycin in vivo Percent Survival Organism Animal: Treated,

Untreated 48 hrs. 96 hrs. 7 days Sm C203Mv -liftiiiin11311 3 8 '3 9 D. pneumomae 1/230 {ifggfi 8 8 8 M. pyogenes var. aureus {Treated 100 100 100 N o. 235. Untreateduhn. 20 20 20 The invention includes within its scope a process for growing the microorganism S. antibioticus. The cultivation of this microorganism preferably takes place in aqueous nutrient media at a temperature of about 24-30 C., and under submerged conditions of agitation and aeration. Nutrient media which are useful for this process include a carbohydrate, such as sugars, starch, glycerol, corn meal, and a source of organic nitrogen, .such as casein, soybean meal, peanut meal, wheat gluten, cottonseed meal, lactalbumin, enzymatic digest of casein, tryptone. A source of growth substances, such as distillers solubles, yeast extract, molasses fermentation residues, as well as mineral salts, such as sodium chloride, potassium phosphate, sodium nitrate, magnesium sulfate and trace minerals such as copper, zinc and iron may also be utilized with desirable results. If excessive foaming is encountered during the fermentation, anti-foaming agents, such as vegetable oils, may be added to the fermentation medium. The pH of the fermentation tends to remain rather constant, but, ifvariations are encountered, a buffering agent, such as calcium carbonate may also be added to the medium.

Inoculum for the preparation of Oleandomycin by the growth of S. antibioticus may be obtained by employing growth from slants on such media as Emersons agar or beef lactose. The growth may be used to inoculate either shaken flasks or inoculum tanks for submerged growth, or, alternatively, the inoculum tanks may be seeded from the shaken flasks. The growth of the microorganism usually reaches its maximum in about two or three days. However, variations in the equipment used, the rate of aeration, rate of stirring and so forth may effect the speed with which the maximum activity is reached. In general, from about 24 hours to four days is a desirable period for producing the antibiotic. Aeration of the medium in tanks for submerged growth is maintained at the rate of about one-half to two volumes of free air per volume of broth per minute. Agitation may be maintained by suitable types of agitators generally familiar to those in the fermentation industry. Aseptic conditions must, of course, be maintained throughout the transfer of the inoculum and throughout the growth of the microorganism.

After growth of the microorganism, the mycelium, which is generally quite luxuriant and fine, may be removed from the fermentation broth by various standard equipment, such as filter-presses, centrifuges, and so forth. The oleandomycin may be recovered from fermentation broth by several different procedures. Alternatively, the whole broth may be used as is or it may be dried. The antibiotic may be further purified by various means; for instance, the compound may be extracted from aqueous solution at neutral or slightly alkaline pHs, preferably between about 6 and about 10, by means of a variety of wator-immiscible organic solvents, including ethers, aromatic hydrocarbons, esters, ketones, lower alcohols and halogenated hydrocarbons. Examples of these are diethyl ether, benzene, toluene, ethyl acetate, butyl acetate, methyl isobutyl ketone, butanol, and chloroform. Even at acidic pHs some of these solvents extract an appreciable amount of antibiotic. This is particularly true of the water-immiscible alcohols, such as butanol, pentanols, and so forth. The antibiotic may be extracted from most solvent solutions back into acidified water, preferably at a pH of below about 2.5. If desired, the solvent extract may be concentrated before extraction into acidified water. By adjustment of the pH to neutrality or alkalinity, the antibiotic may be re-ex'tracted into one of the solvents indicated above. Upon drying the solvent and concentrating the solution the antibiotic crystallizes in long white needles. The product may be recrystallized by cooling a solution thereof in hot ethyl acetate, chloroform, methylene chloride or ethylene dichloride. Further methods of recovery which suggest themselves include absorption on charcoal with subsequent elution, treatment with ion exchange resins, and development on alumina columns.

Oleandomycin is a basic, white, amorphous organic compound that is soluble in dilute, aqueous acids, and moderately soluble in water. It is very soluble in a number of organic solvents, such as methyl alcohol, ethyl alcohol, acetone and butanol. It is insoluble in hexane, carbontetrachlor'ide, and di-n-butyl ether. An aqueous solution of the compound retains its stability for several hours at room temperature over a wide pH range. However, it is quite unstable on heating in acid solution. It is stable in a dry state or dissolved in anyhdrous solvents. The crystalline anhydrous antibiotic hydrochloride has a melting point at about 134-135 C. Both Oleandomycin and its hydrochloride exhibit a broad, low intensity peak in the ultraviolet region at around 286-289 ml mg. in 10 ml. of methanol). When dissolved in chloroform, the antibiotic base shows a number of characteristic peaks in the infrared region, the more significant of which are the following frequencies (in reciprocalv centimeters): 3510, 2910, 2890, 2840, 2790, 1710, 1460, 1385, 1335, 1305, 1280, 1180, 1160, 1110, 1075, 1050, 1005, 985, 960, 935, 895, 885 and 860. The base has been analyzed and found to contain the following elements in the proportions by weight specified:

Carbon 60.36 Hydrogen 9.21 Nitrogen 1.98 Oxygen (by difference) 28.45

When suspended in a potassium bromide pellet, the anhydrous hydrochloride also shows a number of characteristic peaks in the infrared region, the more significant of which are the following frequencies: 3380, 2940, 2890, 1710, 1640, 1465, 1380, 1330, 1190, 1160, 1115, 1075, 1055, 1010, 985, 935, 865, 828. The infrared spectrum is more particularly illustrated in the attached drawings. oleandomycin is designated as amimycin in the drawings. The hydrochloride dissolved in methanol (C, 1%) has an a =-54.

The molecular weight of oleandomycin base, as determined by the ebullioscopic method, was found to be approximately 715.

When a sample of crystalline oleandomycin hydrochloride, which had been crystallized from ethyl acetate was dried at 100 for 18 hours, a loss in weight of 5.0% was observed. The dried material was analyzed and found to contain the following elements in the proportions by weight specified:

Carbon 57.63 Hydrogen 8.73 Nitrogen 1.87 Chlorine (ionic) 4.30 Oxygen (by difference) 27.47 This corresponds to the probable empirical formula C3 aHssNOrzHCl for the anhydrous hydrochloride. The hydrochloride is very soluble in water and also occurs in various hydrated forms, such as the dihydrate, corresponding to the probable empirical formula, CssHeaNOizHCLZHzO.

oleandomycin is clearly distinguished from other antibiotics by its properties, as evidenced by those properties described above and by paper chromatography measurements. Useful salts of the antibiotic can be prepared 1 Example I A slant of S. antibioticus ATCC 11891 on Emerson agar was cultivated under controlled conditions to develop spores for the purpose of inoculating a nutrient medium of the following composition:

Grams Cerelose (dextrose hydrate) 10 Soybean meal 10 Sodium chloride 5 Distillers solubles 5 Calcium carbonate 1 This mixture of nutrients was diluted to a volume of one liter with water, adjusted to a pH of 7 with potassium hydroxide, and subjected to heat sterilization. Thereafter, the medium was cooled and the spores were added thereto under aseptic conditions. The cultivation of the organism was conducted in shaken flasks at about 25 C. for a period of two days. The mixture of broth and mycellum thus formed was then transferred to 20 times its volume of a sterile fermentation medium having the following composition:

Grams/liter Cerelose (dextrose hydrate) 10 Sodium Chloride 5 Curbay Bg (molasses residue) 5 Corn Starch 10 Soybean Meal '10 This medium was adjusted to pH 7 with potassium hydroxide, treated with 1 gram of calcium carbonate per liter, and sterilized in the usual manner, before transferring the broth and mycelium thereto. After seeding the medium with the organism from the shaken flasks, the mixture was subjected to agitation and aeration under sterile conditions for three days. The potency of the broth at this point was found to be meg/ml. The mycelium was removed by filtration and extracted twice with one-quarter volume of methyl isobutyl ketone. The combined solvent phases were concentrated to one-tenth volume under vacuum. The antibiotic was then extracted into water adjusted to a pH of about 2 with sulphuric acid. The aqueous phase was separated, washed with benzene to remove the methyl isobutyl ketone, and adjusted to a pH of 6.5. Thereafter the antibiotic was extracted several times with ether in which it was dried over anhydrous sodium sulfate. Removal of the ether by distillation resulted in crystallization of the antibiotic in the form of long white needles. Finally, the product was recrystallized from hot ethyl acetate. The recrystallized product so obtained was found to be highly eflective against a variety of mycobacteria and Gram-positive microorganisms, as indicated in the tests described above.

Example 11 Another fermentation medium was prepared from the following materials:

Grams Soybean Meal 15 Cerelose (dextrose hydrate) 20 Corn Starch 10 Sodium Chloride 5 Distillers Solubles 5 NZ Amine B (enzymatic digest of casein) 5 These materials were added to one liter of water and the pH of the resulting mixture was adjusted to between 7 and 7.2 with potassium hydroxide. Five grams of calcium carbonate were added to act as a buffer during the fermentation. The medium was then autoclaved and seeded under sterile conditions with S. antibioticus inoculum prepared in accordance with the procedure set forth in Example 1. After subjecting the inoculated medium to aeration and agitation under sterile conditions at about 28 C. for two days, the filtered broth was found to contain micrograms of oleandomycin per ml. of

' solution.

Resort may be had to such modifications and equivalents as fall within the spirit of the invention and the scope of the appended claims.

What we claim is:

1. A process for producing oleandomycin, which comprises cultivating Streptomyces antibioticus No. ATCC 11891 in an aqueous nutrient medium containing a carbohydrate and a source of organic nitrogen, under submerged aerobic conditions until substantial antibacterial activity is imparted to said medium.

2. A process as claimed in claim 1 wherein the oleandomycin is recovered from the fermentation broth.

3. A process for producing oleandomycin, which comprises cultivating Streptomyces antibioticus No. ATCC 11891 in an aqueous nutrient medium containing a carbohydrate and a source of organic nitrogen, under agitated, submerged aerobic conditions at a temperature of from about 24 to about 30 C., for a period of from about one day to about four days.

4. A substance effective in inhibiting the growth of Gram-positive bacteria and mycobacteria, selected from the group consisting of a basic substance moderately soluble in water, very soluble in methyl alcohol, ethyl alcohol, acetone and butanol, insoluble in hexane, carbon tetrachloride and di-n-butyl ether, and capable of forming salts with acids and when dissolved in chloroform exhibits characteristic absorption in the infrared region at the following frequencies expressed in reciprocal centimeters: 3510, 2910, 2890, 2840, 2790, 1710, 1460, 1385, 1335, 1305, 1280, 1180, 1160, 1110, 1075, 1050, 1005, 985, 960, 935, 895, 885 and 860; whose dried crystalline hydrochloride contains the elements carbon, hydrogen, nitrogen, chlorine and oxygen in substantially the following proportions by weight:

Carbon 57.63 Hydrogen 8.73 Nitrogen 1.87 Chlorine (ionic) 4.30 Oxygen (by difierence) 27.47

whose dried crystalline hydrochloride displays a broad, low intensity peak at around 286-289 m in the ultraviolet region of the spectrum, and when dissolved in methanol (C, 1%) has the optical rotation u =54, and when suspended in a potassium bromide pellet exhibits characteristic absorption in the infrared region at the following expressed in reciprocal centimeters: 3380, 2940, 2890, 1710, 1640, 1465, 1380, 1330, 1190, 1160, 1115, 1075, 1055, 1010, 985, 935, 865, 828; and the acid salts of said basic substance.

5. A basic substance effective in inhibiting the growth of gram-positive bacteria and mycobacteria and capable of forming salts with acids; which basic substance is moderately soluble in water, very soluble in methyl alcohol, ethyl alcohol, acetone and butanol, insoluble in hexane, carbon tetrachloride and di-n-butyl ether, and when dissolved in chloroform exhibits characteristic absorption in the infrared region at the following frequencies expressed in reciprocal centimeters: 3510, 2910, 2890, 2840, 2790, 1710, 1460, 1385, 1335, 1305, 1280, 1180, 1160, 1110, 1075, 1050, 1005, 985, 960, 935, 895, 885, and 860; whose dried crystalline hydrochloride contains the elements carbon, hydrogen, nitrogen, chlorine and oxygen in substantially the following proportions by weight:

Carbon 57.63 Hydrogen 8.73 Nitrogen 1.87 Chlorine (ionic) 4.30 Oxygen (by diiference) 27.47

whose dried crystalline hydrochloride displays a broad, low intensity peak at around 286-289 m in the ultraviolet region of the spectrum, and when dissolved in methanol (C, 1%) has the optical rotation oc =-54, and when suspended in a potassium bromide pellet exhibits characteristic absorption in the infrared region at the following frequencies expressed in reciprocal centimeters: 3380, 2940, 2890, 1710, 1640, 1465, 1380, 1330, 1190, 1160, 1115, 1075, 1055, 1010, 985, 935, 865, 828.

6. An acid salt of the basic substance defined in claim 5.

7. A hydrochloride of the basic substance defined in claim 5.

8. The sulfate of the basic substance defined in claim 5.

9. The phosphate of the basic substance defined in claim 5.

References Cited in the file of this patent FOREIGN PATENTS 888,918 Germany July 8, 1949 OTHER REFERENCES Waksman et al.: The Actinomycetes and Their Antibiotics, pub. 1953 by Williams & Wilkins, Baltimore, 

4. A SUBSTANCE EFFECTIVE IN INHIBITING THE GROWTH OF GRAM-POSITIVE BACTERIA AND MYCOBACTERIA, SELECTED FROM THE GROUP CONSISTING OF A BASIC SUBSTANCE MODERATELY SOLUBLE IN WATER, VERY SOLUBLE IN METHYL ALCOHOL, ETHYL ALCOHOL, ACETONE AND BUTANOL, INSOLUBLE IN HEXANE, CARBON TETRACHLORIDE AND DI-N-BUTYL ETHER, AND CAPABLE OF FORMING SALTS WITH ACIDS AND WHEN DISSOLVED IN CHLOROFROM EXHIBITS CHARACTERISTIC ABSORPTION IN THE INFRARED REGION AT THE FOLLOWING FREQUENCIES EXPRESSED IN RECIPROCAL CENTIMETERS: 3510, 2910,
 2890. 2840, 2790, 1710, 1460, 1385, 1335, 1305, 1280, 1180, 1160, 1110, 1075, 1050, 1005, 985, 960, 935, 895, 885, AND 860; WHOSE DRIED CRYSTALLINE HYDROCHLORIDE CONTAINS THE ELEMENTS CARBON, HYDROGEN, NITROGEN, CHLORINE AND OXYGEN IN SUBSTANTIALLY THE FOLLOWING PROPORTIONS BY WEIGHT: 